RU2017146186A - KONOPLI PLANTS WITH MODIFIED THCA-SYNTHASE EXPRESSION - Google Patents

KONOPLI PLANTS WITH MODIFIED THCA-SYNTHASE EXPRESSION Download PDF

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RU2017146186A
RU2017146186A RU2017146186A RU2017146186A RU2017146186A RU 2017146186 A RU2017146186 A RU 2017146186A RU 2017146186 A RU2017146186 A RU 2017146186A RU 2017146186 A RU2017146186 A RU 2017146186A RU 2017146186 A RU2017146186 A RU 2017146186A
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hemp
content
sequence
thca
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Екатерина Александра БУДКО
Томас ШИПЛИ IV
Дуглас Джонсон
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Твид Инк.
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Claims (38)

1. Способ изменения содержания одного или более каннабиноидов в растении конопли, где способ включает даунрегуляцию активности синтазы тетрагидроканнабиноловой кислоты (THCA) в растении конопли.1. A method of changing the content of one or more cannabinoids in a cannabis plant, where the method includes downregulating the activity of tetrahydrocannabinolic acid synthase (THCA) in a hemp plant. 2. Способ по п.1, где изменение содержания каннабиноидов включает уменьшение содержания дельта-9-тетрагидроканнабинола (THC) или увеличение содержания каннабидиола (CBD) в растении конопли.2. The method according to claim 1, where the change in the content of cannabinoids includes reducing the content of delta-9-tetrahydrocannabinol (THC) or increasing the content of cannabidiol (CBD) in a cannabis plant. 3. Способ по п.1, где указанное снижение включает введение в растение выбранной ДНК, кодирующей антисмысловую РНК или миРНК, эффективно подавляющие экспрессию THCA-синтазы, где выбранная ДНК функционально связана с гетерологичным промотором.3. The method of claim 1, wherein said reduction comprises introducing the selected DNA encoding antisense RNA or miRNA into the plant, effectively inhibiting the expression of THCA synthase, where the selected DNA is functionally linked to a heterologous promoter. 4. Способ по п.2, дополнительно включающий отбор растения конопли с требуемым сниженным содержанием THC, повышенным содержанием CBD, или сниженным содержанием THC и повышенным содержанием CBD по сравнению с растением конопли подобного генотипа, которое не включает выбранную ДНК.4. The method of claim 2, further comprising selecting a hemp plant with a desired reduced THC content, increased CBD content, or reduced THC content and increased CBD content compared to a cannabis plant of a similar genotype that does not include the selected DNA. 5. Способ по п.3, где введение выбранной ДНК включает генетическую трансформацию.5. The method according to claim 3, where the introduction of the selected DNA includes genetic transformation. 6. Способ по п.3, где миРНК кодируется:6. The method according to claim 3, where siRNA is encoded: (a) полинуклеотидной последовательностью, обладающей по меньшей мере 85-процентной идентичностью последовательности с последовательностью, выбранной из группы, состоящей из SEQ ID NO:61, 63, 64, 69, 71, 72, 68 и их коплементарной последовательности;(a) a polynucleotide sequence having at least 85% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 61, 63, 64, 69, 71, 72, 68 and their complementary sequence; (b) полинуклеотидной последовательностью, включающей по меньшей мере 21 непрерывный нуклеотид из последовательности, выбранной из группы, состоящей из SEQ ID NO:61, 63, 64, 69, 71, 72, 68 и их коплементарной последовательности; или(b) a polynucleotide sequence comprising at least 21 continuous nucleotide from a sequence selected from the group consisting of SEQ ID NO: 61, 63, 64, 69, 71, 72, 68 and their complementary sequence; or (c) полинуклеотидной последовательностью, включающей по меньшей мере 21 непрерывный нуклеотид THCA-синтазы или их коплементарной последовательности, где THCA обладает по меньшей мере 85-процентной идентичностью последовательности с последовательностью, выбранной из группы, состоящей из SEQ ID NO:1-47.(c) a polynucleotide sequence comprising at least 21 continuous THCA synthase nucleotide or their complementary sequence, where THCA has at least 85% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-47. 7. Способ по п.3, где антисмысловая РНК кодируется:7. The method according to claim 3, where the antisense RNA is encoded: (a) полинуклеотидной последовательностью, включающей SEQ ID NO:81 или ее фрагмент, способной к снижению экспрессии THCA-синтазы; или(a) a polynucleotide sequence comprising SEQ ID NO: 81 or a fragment thereof capable of reducing the expression of THCA synthase; or (b) полинуклеотидной последовательностью, которая по меньшей мере на 85 процентов идентична по меньшей мере 200 непрерывным нуклеотидам из SEQ ID NO:81; и(b) a polynucleotide sequence that is at least 85 percent identical to at least 200 continuous nucleotides of SEQ ID NO: 81; and где транскрипция выбранной ДНК подавляет экспрессию THCA-синтазы.where the transcription of the selected DNA suppresses the expression of THCA synthase. 8. Способ по п.1, где даунрегуляция активности THCA-синтазы включает:8. The method according to claim 1, where the downregulation of the activity of THCA synthase includes: (a) введение в растение конопли или его клетку: (i) по меньшей мере одной РНК-направляемой эндонуклеазы, включающей по меньшей мере один сигнал ядерной локализации, или нуклеиновой кислоты, кодирующей по меньшей мере одну РНК-направляемую эндонуклеазу, включающую по меньшей мере один сигнал ядерной локализации, (ii) по меньшей мере одной направляющей РНК или ДНК, кодирующей по меньшей мере одну направляющую РНК, и, необязательно, (iii) по меньшей мере одного донорного полинуклеотида; и(a) introducing into a cannabis plant or its cell: (i) at least one RNA-directed endonuclease comprising at least one nuclear localization signal, or a nucleic acid encoding at least one RNA-directed endonuclease comprising at least one nuclear localization signal, (ii) at least one directing RNA or DNA that encodes at least one directing RNA, and, optionally, (iii) at least one donor polynucleotide; and (b) культивирование растения конопли или его клетки таким образом, что каждая направляющая РНК направляет РНК-направляемую эндонуклеазу к целевому участку в хромосомной последовательности, где РНК-направляемая эндонуклеаза вводит двухцепочечный разрыв в целевой участок, и двухцепочечный разрыв восстанавливается в процессе репарации ДНК, таким образом, что хромосомная последовательность изменяется, где целевой участок расположен в гене THCA-синтазы, и хромосомная модификация прерывает или нарушает транскрипцию и/или трансляцию гена THCA-синтазы.(b) cultivating the cannabis plant or its cells in such a way that each guide RNA directs the RNA-directed endonuclease to the target site in the chromosomal sequence, where the RNA-directed endonuclease introduces a double-stranded break to the target site, and the double-stranded break is restored during the DNA repair process, such the chromosomal sequence is changed, where the target region is located in the THCA synthase gene, and the chromosomal modification interrupts or disrupts the transcription and / or translation of the THCA gene intazy. 9. Способ по п.8, где РНК-направляемая эндонуклеаза получена из кластерных регулярно расположенных коротких палиндромных повторов (CRISPR)/CRISPR-ассоциированной (Cas) системы.9. The method of claim 8, where the RNA-directed endonuclease is obtained from cluster regularly spaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system. 10. Способ по п.8, где даунрегуляция активности THCA-синтазы не встраивает экзогенный генетический материал и приводит к получению не существующего в природе растения конопли или его клетки.10. The method of claim 8, where the downregulation of the activity of THCA synthase does not embed exogenous genetic material and results in a cannabis plant that does not exist in nature or its cells. 11. Способ по п.1, где даунрегуляция активности THCA-синтазы включает:11. The method according to claim 1, where the downregulation of the activity of THCA synthase includes: (a) идентификацию по меньшей мере одного локуса гена THCA-синтазы в последовательности ДНК в растении конопли или его клетки;(a) identifying at least one locus of the THCA synthase gene in a DNA sequence in a cannabis plant or its cell; (b) идентификацию по меньшей мере одной специфичной последовательности распознавания эндонуклеазы по меньшей мере в одном локусе гена THCA-синтазы;(b) identification of at least one specific endonuclease recognition sequence in at least one locus of the THCA synthase gene; (c) введение в растение конопли или его клетку по меньшей мере первой сконструированной эндонуклеазы, где растение конопли или его клетка включают последовательность распознавания для сконструированной эндонуклеазы в или вблизи от локуса гена THCA-синтазы, и сконструированная эндонуклеаза транзиентно или стабильно экспрессируется;(c) introducing into the hemp plant or its cell at least the first designed endonuclease, where the hemp plant or its cell includes a recognition sequence for the designed endonuclease at or near the THCA synthase gene locus, and the designed endonuclease is transiently or stably expressed; (d) анализ растения конопли или его клетки на наличие опосредованной сконструированной эндонуклеазой модификации в ДНК, составляющей или фланкирующей локус гена THCA-синтазы; и(d) analyzing a hemp plant or its cell for the presence of a mediated endonuclease-engineered modification in the DNA that constitutes or flank the locus of the THCA synthase gene; and (e) идентификацию растения конопли, его клетки или клетки его потомства как включающих модификацию в локусе гена THCA-синтазы.(e) the identification of the cannabis plant, its cells or the cells of its progeny as including a modification in the locus of the THCA synthase gene. 12. Трансгенное растение конопли, полученное способом по п.1.12. Transgenic hemp plant, obtained by the method according to claim 1. 13. Семя трансгенного растения по п.‎12, где семя включает выбранную ДНК.13. The seed of the transgenic plant according to claim 12, wherein the seed includes the selected DNA. 14. Растение конопли с пониженным содержанием дельта-9-тетрагидроканнабинола (THC), повышенным содержанием каннабидиола (CBD) или пониженным содержанием THC и повышенным содержанием CBD, включающее пониженную экспрессию синтазы тетрагидроканнабиноловой кислоты (THCA).14. Cannabis plant with reduced delta-9-tetrahydrocannabinol (THC) content, increased cannabidiol content (CBD) or low THC content, and increased CBD content, including reduced expression of tetrahydrocannabinolic acid synthase (THCA). 15. Растение конопли по п.14, включающее выбранную ДНК, кодирующую антисмысловую РНК или миРНК, эффективно подавляющие экспрессию THCA-синтазы, где выбранная ДНК функционально связана с гетерологичным промотором, где растение конопли включает пониженное содержание THC, повышенное содержание CBD, или пониженное содержание THC и повышенное содержание CBD по сравнению с растением конопли подобного генотипа, которое не включает выбранную ДНК.15. The cannabis plant of claim 14, comprising selected DNA encoding antisense RNA or miRNAs that effectively suppress the expression of THCA synthase, where the selected DNA is functionally linked to a heterologous promoter, where the cannabis plant includes reduced THC content, increased CBD content, or reduced content THC and increased CBD levels compared to a hemp plant of a similar genotype that does not include the selected DNA. 16. Семя, клетка или часть растения конопли по п.14.16. The seed, cell or part of the hemp plant of claim 14. 17. Продукт из растения конопли, полученный из растения по п.14.17. The product of the hemp plant, derived from the plant of claim 14. 18. Продукт из растения конопли по п.17, включающий суммарную концентрацию каннабидиоловой кислоты и каннабидиола от приблизительно 18% до приблизительно 60% или от приблизительно 7% до приблизительно 30% по весу.18. The product of the cannabis plant of claim 17, comprising a total concentration of cannabidiolic acid and cannabidiol from about 18% to about 60% or from about 7% to about 30% by weight. 19. Продукт из растения конопли по п.17, включающий суммарную концентрацию дельта-9-тетрагидроканнабинола и тетрагидроканнабиноловой кислоты от приблизительно 0% до приблизительно 3% по весу.19. A product from the hemp plant of claim 17, comprising a total concentration of delta-9-tetrahydrocannabinol and tetrahydrocannabinolic acid from about 0% to about 3% by weight. 20. Продукт из растения конопли по п.17, включающий конопляное масло, настойку конопли, высушенные цветки конопли и/или высушенные листья конопли.20. Product from the hemp plant of claim 17, including hemp oil, hemp tincture, dried hemp flowers, and / or dried hemp leaves. 21. Продукт из растения конопли по п.17, адаптированный для ингаляции, употребления внутрь, подъязычного применения или наружного применения.21. Product from the hemp plant of claim 17, adapted for inhalation, ingestion, sublingual use, or topical use. 22. Способ получения медицинской композиции на основе конопли, где способ включает22. The method of obtaining a medical composition based on hemp, where the method includes (a) получение растения конопли по п.14;(a) obtaining a hemp plant of claim 14; (b) выращивание растения конопли при условиях роста растения, с получением растительной ткани из растения конопли; и(b) growing a cannabis plant under growth conditions of a plant, to obtain plant tissue from a hemp plant; and (c) изготовление медицинской композиции на основе конопли из растительной ткани.(c) the manufacture of a hemp-based medical composition from plant tissue.
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